Various publications, including patents, published applications and scholarly articles, are cited throughout the specification. Each of these publications is incorporated by reference herein, in its entirety. Citations not fully set forth within the specification may be found at the end of the specification.
Coffee aroma and flavor are key components in consumer preference for coffee varieties and brands. The characteristic aroma and flavor of coffee stems from a complex series of chemical reactions involving flavor precursors (Maillard reactions) that occur during the roasting of the bean. Flavor precursors include chemical compounds and biomolecules present in the green coffee bean. To date, over 800 chemicals and biomolecules have been identified as contributing to coffee flavor and aroma. (Flament, I., 2002 “Coffee Flavor Chemistry” J. Wiley U.K.). Because coffee consumers are becoming increasingly sophisticated, it is desirable to produce coffee with improved aroma and flavor in order to meet consumer preferences. Both aroma and flavor may be artificially imparted into coffee products through chemical means. See, for example, U.S. Pat. No. 4,072,761 (aroma) and U.S. Pat. No. 3,962,321 (flavor). However, to date, there is little information concerning the influence of natural coffee grain components such as polysaccharides, proteins, pigments, and lipids, on coffee aroma and flavor. One approach is to select varieties from the existing germplasm that have superior flavor characteristics. A disadvantage to this approach is that, frequently, the highest quality varieties also possess significant negative agronomics traits, such as poor yield and low resistance to diseases and environmental stresses. It is also possible to select new varieties from breeding trials in which varieties with different industrial and agronomic traits are crossed and their progeny are screened for both high quality and good agronomic performance. However, this latter approach is very time consuming, with one crossing experiment and selection over three growing seasons taking a minimum of 7-8 years. Thus, an alternative approach to enhancing coffee quality would be to use techniques of molecular biology to enhance those elements responsible for the flavor and aroma that are naturally found in the coffee bean, or to add aroma and flavor-enhancing elements that do not naturally occur in coffee beans. Genetic engineering is particularly suited to achieve these ends. For example, coffee proteins from different coffee species may be swapped. In the alternative, the expression of genes encoding naturally occurring coffee proteins that positively contribute to coffee flavor may be enhanced. Conversely, the expression of genes encoding naturally occurring coffee proteins that negatively contribute to coffee flavor may be suppressed.
Coffees from different varieties and origins exhibit significant flavor and aroma quality variations when the green grain samples are roasted and processed in the same manner. The quality differences are a manifestation of chemical and physical variations within the grain samples that result mainly from differences in growing and processing conditions, and also from differences in the genetic background of both the maternal plant and the grain. At the level of chemical composition, at least part of the flavor quality can be associated with variations in the levels of small metabolites, such as sugars, acids, phenolics, and caffeine found associated with grain from different varieties. It is accepted that there are other less well characterized flavor and flavor-precursor molecules. In addition, it is likely that structural variations within the grain also contribute to differences in coffee quality. One approach to finding new components in the coffee grain linked to coffee quality is to study the genes and proteins differentially expressed during the maturation of grain samples in different varieties that possess different quality characteristics. Similarly, genes and proteins that participate in the biosynthesis of flavor and flavor-precursor molecules may be studied.
The flavonoids form a large group of ubiquitous plant secondary metabolites, with over 4000 molecules of this class identified to date (Bovy et al. (2002) and Yilmaz et al. (2004)). Flavonoids are derived from the condensation of p-coumaroyl-CoA, which is synthesized from phenylalanine via the early phenylpropanoid pathway, and three molecules of malonyl-CoA, which is generated by the TCA cycle. (Dixon et al. (1999); Winkel-Shirley (2002); and Dixon (2005)). The various flavonoid metabolites contribute in different ways to the normal functioning and survival of the plant. For example, the red, blue and purple anthocyanin pigments found in flowers participate in plant reproduction by their involvement in attracting insects for pollination. (Winkel-Shirley (2002)). Other flavonoids, the brown proanthocyanidin pigments (also termed condensed tannins), are believed to have antimicrobial properties and thus have been proposed to contribute to microbial resistance. (Sivakumaran et al. (2004); and Cos et al. (2005)). Yet another group of flavonoids, the isoflavones which are synthesised primarily in leguminous plants, are involved in plant-microbe interactions. (Dixon (2005)). For example, isoflavones are continuously excreted from the roots of legumes and molecules such as daidzein have been shown to induce nodulation related genes in the nodulating Rhizobium bacteria (Kobayashi et al. (2004)).
In recent years, an increasing number of studies have focused on the relationship between plant-derived foods containing flavonoids and human health. Both academic and applied interest in this area is stimulated by the fact that some widely consumed plant foods are relatively rich in flavonoid/phenolic compounds and by the fact that people who consume higher quantities of these foods appear to have lower risks for certain significant heath problems, such as cardiovascular disease and cancer. (Bazzano et al. (2002); Clifford (2004); Cos et al. (2005); and Go et al. (2005)). The importance of flavonoids to human and animal health is supported by detailed experimental data, which indicate that flavonoids can have specific functional interactions within mammalian cells. For example, the antioxidant properties of flavonols, such as kaempferol and quercetin, have been broadly shown to give some protection against oxidative stress. (Sugihara et al. (1999); and Duthie et al. (2000)). Daily oral administration of the flavonoid quercetin has been shown to exhibit both anti-hypertensive and antioxidative effects in hypertensive rats. (Garcia-Saura et al. (2005)).
The flavonoids present in dark chocolate are currently being intensively studied, and a recent study showed that consumption of dark chocolate rich in flavonoids may lower blood pressure, presumably through the ability of one or more of the flavonoids to increase nitric oxide bioavailability. (Grassi et al. (2005)). Resvertrol is another flavonoid related molecule that is currently of interest. This phytoalexin is found in grapes and other foods, and has been found to be active as a cancer chemoprevention agent, (Jang et al. (1997)), and to have the potential to delay aging via its ability to activate Sir-2 like proteins (Sirtuins). (Wood et al. (2004)). Higher dietary intake of other flavonoids, like the isoflavonoids found in soy, has also been associated with reduced levels of cancer, (Setchell et al. (1999)), and dietary intake of the isoflavone genistein has been shown to reduce the susceptibility of rats to mammary cancer, (Lamartiniere et al. (2002), and helps prevent bone loss caused by estrogen deficiency in female mice. (Ishimi et al. (1999)).
The early steps of the plant phenylpropanoid pathway leading to the key flavonoid precursors p-coumaryl-CoA have been described in several plants (FIG. 1A; Dixon et al. (1995); and Winkel-Shirley (2002). The first step in the phenylpropanoid pathway is the deamination of phenylalanine to cinnamic acid by L-phenylalanine ammonia lyase (PAL). Four different PAL genes have been characterized in Arabidopsis and these appear to fall into two different groups. (Raes et al. (2003)). As expected for a major branch-point between the plant primary and secondary metabolic pathways, the expression and activities of the different PAL isoforms are under complex regulatory control. (Dixon et al. (1995); and Rohde et al. (2004)). The next enzyme in the pathway is the protein trans cinnamate-4-hydroxylase (C4H; CYP73A5). Only one gene has been found for this P450-dependent mono-oxygenase in arabidopsis, while in some other plants, two or more C4H genes can been found that fall into two distinct classes. (Raes et al. (2003)). The next step, the production of p-coumaryl-CoA, is carried out by 4-coumarate:CoA ligase (4CL). In the arabidopsis genome, there are at least four 4CL genes and nine 4CL-like genes. (Raes et al. (2003)). In addition to forming p-coumaryl CoA, the 4CL proteins characterized from arabidopsis, as well as the characterized 4CL proteins from soybean we found to be capable of forming CoA esters with caffeic acid and ferulic acid at different efficiencies, and the At4CL4 protein of arabidopsis and the Gm4CL1 protein of soybean were also found to be capable of forming CoA esters with 5-hydroxyferulic acid and sinapic acid. (Hu et al. (1998); Lindermayr et al. (2002); Schneider et al. (2003); and Hamberger et al. (2004)).
A number of recent reviews on the core flavonoid synthesis pathway have been published (Winkel-Shirley (2002) and Dixon (2005), and the current understanding of this pathway is outlined schematically in FIG. 1B. (Winkel-Shirley (2002), and Xie et al. (2004)). The first step of this pathway, which is catalyzed by chalcone synthase (CHS), is the condensation of p-coumaryl CoA with three molecules of malonyl CoA to form tetrahydroxychalcone (naringenin chalcone). In some plants, particularly the leguminous plants, the enzyme chalcone reductase (CHR) can also be present (FIG. 1B). This enzyme is thought to act on an intermediate of the CHS multistep reaction, and the CHS/CHR coupled reaction is proposed to yield chalcone (4,2′,4′,6′-tetrahydroxychalcone) and deoxychalcone (4,2′,4′-trihydroxychalcone). (Bomati et al. (2005)). These CHS/CHR products are then precursors for a group of phytoalexins that are often produced in response to herbivore and pathogen attacks, and for the synthesis of CHR derived products that are involved in symbiotic root nodulation by nitrogen fixing Rhizobium bacteria. (Dixon et al. (1999)).
In the core flavonoid pathway, the product of CHS (naringenin chalcone) is transformed into (2S)-5,7,4′-trihydroxyflavanone (naringenin) by chalcone isomerase (CHI). Two types of CHI have been found, with type I being ubiquitous in the plant kingdom, while the type II CHI, which has a broader substrate range, appears to be most frequently found in leguminous plants. (Ralston et al. (2005)). The next reaction in the pathway is the addition of a hydroxyl group at the C3 position of the C ring to form 2,3-dihydrokaempferol (DHK) and is catalyzed by F3betaH, a 2-oxoglutarate dependent dioxygenase. (Dixon et al. (1999); Wellman et al. (2004); and Dixon (2005)). As indicated in FIG. 1B, DHK can be further hydroxylated at the 3′ and 5′ positions of the B ring by the P450 dependent enzymes F3′H and F3′5′H forming 2,3-dihydroquercetin (DHQ) and 2,3dihydromyricetin (DHM) respectively. Dihydroflavonol-4-reductase (DFR) catalyses the next reaction, the addition of a hydroxyl group at the 4 position of ring C of DHK, DHQ, and DHM (synthesized by the F3H family of enzymes) to yield leucopelargonidin, leucocyanidin or leucodelephinidin respectively. However, some plant DFR proteins do not accept the monohydroxylated DHK and thus these plants may not be able to make the associated downstream products. (Johnson et al. (2001)). Only one DFR gene has been found in plants such as Arabidopsis and tomato, and interestingly, in plants with several DFR genes, it appears that only one of these genes produces an active protein. (Xie et al. (2004)). The products of DFR are key precursors for the synthesis of the anthocyanins and condensed tannins and it has been noted that herbivore attack induces DFR expression in plants. Furthermore, this induction is associated with an increase in the synthesis of condensed tannins, a group of molecules that have been implicated in protecting plants from herbivores. (Peters et al. (2002)). Immediately downstream of DFR, the enzyme anthocyanidin synthase (ANS; leucoanthocyanidin dioxygenase) is capable of forming anthocyanidins from the different DFR products (i.e., from the leucoanthocyanins), and these ANS products can be subsequently glycosylated to form the anthocyanins. In addition, leucoanthocyanidin reductase (LAR) can also convert the leucoanthocyanidins to form 2,3-trans-flavan-3-ols (catechins). The related 2,3-cis-flavan-3-ols (epicatechins) are formed via the action of ANS and anthocyanidin reductase (ANR) which uses NADPH to reduce anthocyanidins. (Xiet et al. (2003)). Finally, there is little currently known about the last step(s) involved in the formation of condensed tannins from trans- and cis-flavan-3-ol monomers.
It is well known that flavonoids, and related glycosylated derivatives, make significant flavor contributions to beverages produced from plant ingredients. For example, grapefruit citrus are known to have a bitter flavor, which is in part due to the presence of a flavanone (flavanone-7-neohesperidosides), while oranges, in contrast, are generally less bitter, having only tasteless flavanone-7-rutinosides. (Frydman A et al. (2004). Likewise, it is well known that the flavonoids and related molecules in grapes contribute significantly to the astringency and bitterness characteristics of different wines (Monagas M et al. (2005)). Finally, fruit juices and other beverages such as tea contain anthocyanidins at levels that contribute significantly to the flavor and astringency of these beverages. (Dixon R et al. (2005b); and, Lesschaeve I et al. (2005)). The anthocyanidins are monomers, oligomers and polymers of molecules produced by the flavonoid pathway. Considering the observations above, it can thus be expected that by altering the levels, and/or the molecular profiles of the precursors, or by altering the polymerization levels and profiles of the final products in the starting plant material, it could be possible to alter the flavor and astringency profiles of beverages made from these raw materials.
There is currently little information published on the presence of flavonoids in the green or roasted coffee grain and whether these molecules or derivatives thereof contribute to the flavor of coffee. However, there is one recent report that suggests that flavonoids are present in roasted coffee. (Yen et al. (2005)). It is noted, however, that the method used by these investigators to determine flavonoid content is a generalized total flavonoid method and thus could provide an artificially inflated measurement of this broad class of molecules in roasted coffee. Accordingly, more detailed work is required to examine the flavonoids present in both the coffee grain and in the roasted product. One study has recently been carried out that begins to address the flavonoids present in the fruit part of the coffee cherry (pericarp). It has been found that ripe coffee arabica cherry fruit contains three major classes of flavonoids: the flavan-3-ols (monomers and procyanidins), flavonols, and anthocyanidins. (Ramirez-Coronel et al. (2004)). Given the known roles these compounds play in other plants, it can be presumed that the coffee flavonoids are also involved in protecting the fruit tissues from UV and oxidation related stresses, and in protecting the cherries from microbial and insect attack. Because of the health benefits of flavonoids in the human diet, and because these molecules also have agronomic benefits (herbivore/pathogen and stress resistance), it is of interest to examine the flavonoid pathway in coffee.
From the foregoing discussion, it will be appreciated that modulating flavonoid content in coffee grain by genetically modulating the production of the proteins responsible for early phenylpropanoid and flavonoid biosynthesis would be of great utility to enhance the aroma and flavor of coffee beverages and coffee products produced from such genetically engineered coffee beans. Enhanced flavonoid content and/or altered flavonoid profile in the coffee bean may also positively contribute to the overall health and wellness of consumers of coffee beverages and products produced from such coffee beans. In addition, modulating flavonoid content in the coffee plant has implications for protecting the coffee fruit from ultraviolet, oxidative, microbial, or insect stress or damage. Accordingly, a need exists to identify, isolate and utilize genes and enzymes from coffee that are involved in the biosynthesis of early phenylpropanoids and flavonoids.